Identification of ADRA2A Polymorphisms Related to Shear-Mediated Platelet Function by the PFA-100® System.

[Background]

α 2A adrenergic receptor (ADRA2A) on platelets interacts with epinephrine, which has a key role in regulating platelet functions. There is familial clustering of inter-individual variations in the epinephrine-induced platelet aggregation, the molecular basis of which, however, has not been fully understood. In this study, we screened the sequence variations in the transcriptional region of ADRA2A gene and analyzed the relationship between the two common polymorphisms and platelet function using collagen/epinephrine (CEPI) cartridge in the platelet function analyzer (PFA)-100® system.

[Methods and Results]

Written informed consent was obtained from all study subjects who were genetically unrelated healthy Japanese males (n=255). Of the 255 subjects, an initial screening for the sequence variation(s) in the ADRA2A transcriptional region was performed on 44 subjects enrolled in 2003. Subsequently, relationship between ADRA2A polymorphisms and platelet function was investigated in 211 subjects enrolled between 2004 and 2005. In the screening, we observed 16 single nucleotide polymorphisms (SNPs) including 5 novel variations. We next examined the association between the ADRA2A polymorphisms and epinephrine-mediated platelet function using the PFA-100® system under high shear conditions (5000–6000/sec). According to the pilot study on the 16 SNPs, we focused on 1780A/G and 2732A/G (the first nucleotide of the open reading frame is nt#1, which corresponds to nt#31586326 of the GenBank NT_030059). We measured platelet function, as assessed by closure time (CT) using CEPI cartridge. A longer CEPI-CT indicates lower platelet function in the interaction with collagen and epinephrine. Because the manufacturer’s instructions for the PFA-100® report a mean CEPI-CT value of 132 s, study subjects were divided in two groups: a higher function group with a CEPI-CT < 132 s (n=90) and a lower function group with a CEPI-CT ≥ 132 s (n=121). The frequency of the 1780GG genotype in the lower function group was significantly higher than that in the higher function group (p=0.0478) whereas no association between the CEPI-CT and the 2372A/G polymorphism was observed (p=0.1164). We also observed the effect of the combination of 1780GG+2372AA genotypes for shorter CT (p=0.0319). A multiple logistic regression model to adjust the reported confounding factors (VWF: RCo, platelet count, and hematocrit) revealed an adjusted odds ratio of 2.10 (p=0.0274) for the genotypic combination or 1.82 (p=0.0757) for the 1780A/G alone. Plasma VWF:RCo level was also an independent predictor for CEPI-CT. These observations suggested the enhanced effect of the genotypic combination as compared with the 1780A/G alone.

[Conclusion]

Of the 16 sequence variations of the transcriptional region of the ADRA2A gene, the combination of the 1780A/G and 2372A/G polymorphism was associated with collagen/epinephrine-mediated platelet function by PFA-100® system.